Cloning of ERAP1 haplotypes has been described (26 (link)). For expression of short antigenic peptides, forward and reverse oligonucleotides were annealed and ligated into pcDNA3.1D/V5-His-TOPO® vector using the Directional TOPO Expression kit (Invitrogen) as described (22 (link)).
To compare ERAP1 Hap2 and Hap10 activity, ERAP1-/- WM793 clones were transfected with pcDNA-ERAP1 Hap2 or Hap10 (100 ng) at 60-80% cell density using FuGENE HD (Promega). 24 h after transfection, HLA expression was evaluated, or Vα3S1/Vβ13S1-TCR hybridoma cells were added and assessed for sGFP induction after 24 h of co-culture by flowcytometry. Parental or ERAP1-/- HEK293T cells were seeded on 48 well plates and co-transfected with pRSV-HLA-C*06:02 (75 ng), plasmid encoded ADAMTSL5 peptides (75 ng) and either pcDNA-ERAP1 Hap2, Hap10, or pcDNA-vector (75 ng) using FuGENE HD. 24 h later, Vα3S1/Vβ13S1-TCR hybridoma cells were added. Staining with CD8-PerCPCy5.5 (Biolegend #344710) differentiated hybridoma cells from ERAP1-/- HEK293T cells. Gating strategy to examine TCR hybridoma stimulation is given inSupplemental Fig. 2A .
To compare ERAP1 Hap2 and Hap10 activity, ERAP1-/- WM793 clones were transfected with pcDNA-ERAP1 Hap2 or Hap10 (100 ng) at 60-80% cell density using FuGENE HD (Promega). 24 h after transfection, HLA expression was evaluated, or Vα3S1/Vβ13S1-TCR hybridoma cells were added and assessed for sGFP induction after 24 h of co-culture by flowcytometry. Parental or ERAP1-/- HEK293T cells were seeded on 48 well plates and co-transfected with pRSV-HLA-C*06:02 (75 ng), plasmid encoded ADAMTSL5 peptides (75 ng) and either pcDNA-ERAP1 Hap2, Hap10, or pcDNA-vector (75 ng) using FuGENE HD. 24 h later, Vα3S1/Vβ13S1-TCR hybridoma cells were added. Staining with CD8-PerCPCy5.5 (Biolegend #344710) differentiated hybridoma cells from ERAP1-/- HEK293T cells. Gating strategy to examine TCR hybridoma stimulation is given in
